DNA and other molecular level analysis can detect genetic variations, which comprise single nucleotide polymorphisms (SNPs) and structural variations (SVs). These can be further divided into microscopic (larger than 3 Mb) and submicroscopic variations. SVs can be defined as all genomic changes that are not single base-pair substitutions and include insertions, deletions, inversions, duplications and translocations of DNA sequences, as well as copy number variants (CNVs). Starting in the mid 1980s, the process of DNA extraction, testing, and analysis was laborious and required months of time utilizing specialized equipment in research laboratories to generate and analyze the data. With the advent of the polymerase chain reaction (PCR), the process for obtaining DNA-based information became faster and more efficient. While PCR technology has greatly simplified the process, DNA analysis today is performed by highly trained technicians in either research or clinical laboratories. The process generally requires the extraction of the DNA from the biological sample followed by PCR amplification. The extraction step can be accomplished using an automated DNA extractor, a highly specialized piece of equipment, which can be bench top-sized or larger. Manufacturers of these devices include Autogen, Invitrogen, and Promega. The extraction step can also be done manually by a technician using a kit (e.g., a Qiagen kit). Each sample must be extracted separately, and the manual extraction step is a source of potential contamination of the sample. Once the DNA has been extracted, the DNA is amplified using a thermal cycler or thermal cycler as a separate piece of equipment from the extractor. Manufacturers of thermal cyclers include Eppendorf, Applied Biosystems, BioRad, and Hitachi-300. Detection of PCR products is usually accomplished via fluorescence. Once detection has occurred, there needs to be an additional step to analyze and interpret the results for clinical relevance. For example, the relation of single nucleotide polymorphisms (SNPs) in a DNA sample to a predicted drug response for a patient requires a detailed bioinformatic analysis step, which often can take 14 days or longer for results to be received.
In addition to PCR, there are a number of other technologies for DNA analysis, all of which require highly trained technicians in a clinical or research laboratory setting utilizing specialized equipment. Microarray technology provides new analytical devices that allow the parallel and simultaneous detection of several thousands of probes within one sample. Microarrays, sometimes called DNA chips, are widely used in gene expression analysis, genotyping of individuals, analysis of point mutations and singlenucleotide polymorphisms (SNP), as well as other genomic or transcriptomic variations. For microarray technologies, a separate device for detection is needed (instead of a thermal cycler) to detect fluorescence. Different types of microarrays include printed arrays, in situ-synthesized oligonucleotide arrays (includes Roche NimbleGen, Affymetrix GeneChip, and Agilent), high-density bead arrays (includes Illumina BeadArray), electronic arrays (includes Nanogen NanoChip), and suspension bead arrays (includes Luminex xTAG). Microarrays are plagued by false positives and questionable quantifications, and the data require a separate and complex analysis step. Additionally, DNA microarrays have some problems in terms of reproducibility and reliability due to the fact that the DNA probes are fixed on electrodes.
Another technique is fluorescence in situ hybridization (FISH). FISH allows the mapping of specific DNA sequences at high resolution. However, it is time-consuming and labor-intensive, which limits its application as a genome-wide variation screening tool. FISH can be used to detect microscopic structural variations larger than 3 Mb, including visible chromosomal heteromorphisms, reciprocal translocations, deletions, duplications, insertions and inversions.
More recently, lab-on-a-chip technologies are compact in size and enable low sample volumes (nanoliter) and short analysis time (less than 10 sec to complete one PCR cycle, 370 sec for completing the whole quantification process). Some disadvantages are detection limits, quantification uncertainties, and melting analysis ability of chip prototypes. Other technologies include multiplex amplifiable probe hybridization (MAPH) and multiplex ligation-dependent probe amplification (MLPA), which can efficiently detect the specific changes at 50-100 genomic loci in a single experiment. MAPH is fast and cost-effective in detecting small genomic changes, but the limited multiplicity owing to gel-based detection is a major drawback. MAPH combined with microarrays increases the detection throughput.
PCR microfluidics enables large numbers of parallel amplification analyses on a single chip and can produce more accurate information and greater understanding necessary for some particular bioassays, which, however, are difficult, unpractical, or even impossible to perform on a macro-scale PCR device. Besides, single molecule PCR can be easily performed in PCR microfluidics, starting with a single-copy sequence in the PCR mixture. Much smaller PCR vessels can increase resolution while reducing the overall size of the PCR device, but effects related to the non-specific adsorption of biological samples to the surfaces of the vessel may become significant as a result of the increased Surface-to-Volume Ratio (SVR) upon miniaturization, which may inhibit PCR amplification. As is seen from the development history of PCR microfluidics, another “bottleneck” blocking the realization of a truly integrated DNA analyzer may be a portable detection module for on-line PCR product detection. The most common detection scheme is off-line or on-line CE separation of the PCR product, usually followed by laser induced fluorescence detection or in some cases by EC detection. However, optical detection systems are difficult to miniaturize onto a monolithic microanalytical system. Furthermore, the electrophoretic separation and detection technique cannot provide data on the sequence of the PCR product since it mainly serves to separate DNA fragments of different sizes from a mixture of DNA fragments. To acquire information concerning the sequence of a PCR product, the DNA microarray hybridization, which is a sequence-based detection method, has been integrated into PCR microfluidics platforms. However, the use of DNA microarrays has some problems in terms of reproducibility and reliability due to the fact that the DNA probes are fixed on electrodes. [1]
Overall, gel-based genotyping assays such as PCR-restriction fragment length polymorphism (RFLP) analysis, oligonucleotide ligation assay genotyping, and mini-sequencing are relatively straightforward and are useful when dealing with a small number of samples. The methods are labor-intensive and require experienced and skilled technical staff for final analysis. Although gel-based genotyping methods are still widely used in many laboratories, they are difficult to apply to high-throughput genotyping in large-scale pharmacogenetic studies. [2]
Next Generation Sequencing (NGS) has made significant strides in the past few years. Three NGS technologies available are Roche 454, ABI SOLiD, and Illumina. These technologies vary considerably in terms of throughput, read-lengths, and cost, which is in the thousands of dollars per sample. Both the Illumina and Roche 454 platforms share the underlying principle of ‘sequencing by extension’ used in the Sanger methodology (single bases complementary to the template molecule are sequentially added to a nascent strand and their identity determined by chemical means). The ABI sequencing technology uses a unique chemistry whereby oligonucleotides complementary to a series of bases in the sequencing template are ligated to a nascent molecule and the identity of the first two bases of the ligated oligonucleotide is specified by a degenerate four color code (each color specifies four different dinucleotides).